Project Summary/Abstract Messenger ribonucleic acid (mRNA) plays key roles in cells and organisms as a carrier of protein-coding information and as a regulator of gene expression. The pharmaceutical industry has begun to exploit the many natural functions of mRNA to develop novel therapeutics and vaccines, which promise high efficacy and great flexibility in the prevention and treatment of diseases ranging from cancers and infectious diseases such as hepatitis and HIV to genetic diseases such as cystic fibrosis and rare diseases caused by heritable genetic defects. The expanded effort in mRNA therapeutics and mRNA vaccines has created a new demand for mRNA molecules manufactured in large quantities to precise specifications. In particular, the need to create mRNA molecules >1kb in length that are free of unwanted side products, and to incorporate modified nucleotides for more efficient delivery, higher stability and better clinical efficacy, has compounded this manufacturing problem. Although RNAs can be produced enzymatically in vitro with the use of specialized RNA polymerases, the enzymes widely used to produce RNA for R&D purposes are not suited for the demanding specifications that apply to RNA molecules intended for mRNA therapeutics and vaccines. A new class of enzymes, highly optimized for synthesis of long RNAs with specific sequences and structures, need to be created to meet this new demand. In a Phase I feasibility project, Primordial Genetics discovered and tested 53 novel RNA polymerases of which 13 were found to be superior to the current enzymes used for RNA manufacturing. On the strength of our Phase I results, the company began to forge connections to RNA therapeutics companies who are very interested in testing our new enzymes. In the proposed Phase II project, we will improve four of these enzymes, characterize the activity of six improved enzymes in detail, and prepare methods and datasets for using these enzymes for clinical mRNA manufacturing. Our goal is to create enzymes that can meet the varied needs for manufacturing a diversity of mRNA sequences, sizes and chemical structures represented in mRNA vaccines and mRNA therapeutic products under development. The enzymes discovered and improved in this work will be directly useful for mRNA manufacturing applications, and will be licensed or sold to companies developing mRNA vaccines and therapeutics as well as companies building RNA manufacturing capabilities.